The hairpin ribozyme: discovery, two-dimensional model, and development for gene therapy.

This review chronicles the discovery of the hairpin ribozyme, its characterization, and determination of the two-dimensional structure, culminating with its use for human gene therapy as an AIDS therapeutic. The minimal sequence constituting the hairpin ribozyme catalytic domain was identified from a small plant viral satellite RNA. Biochemical characterization showed it to be among the most efficient of all known ribozymes. Mutagenesis determined that the two-dimensional structure had four helices, consisting of 17 Watson-Crick base pairs and one A:G pair for a total of 18 bp. The helices were interspersed with five single-stranded loops. Helices 1 and 2 were located between the ribozyme and substrate, allowing the ribozyme to recognize the substrate. The substrate had a sequence preference of BNGUC where * is the site of cleavage and NGUC the substrate loop between these two helices. By using sequences of this type, it was possible to design the ribozyme to base pair with the substrate and cleave heterologous RNA substrates-leading to design of the hairpin ribozyme for gene therapy. The HIV-1 sequence was searched for suitable target sites, and ribozymes were designed, optimized, catalytically characterized, and tested in vivo against HIV-1 targets. Two ribozymes had excellent in vitro catalytic parameters and inhibited in vivo expression of viral proteins by 3-4 logs in tissue culture cells. Viral replication was inhibited as well. They have been developed as human AIDS therapeutics, and will likely be the first ribozymes to be tested as human drugs in clinical trials.